胆碱能M受体调控细胞周期蛋白依赖性激酶5及其在敌敌畏诱导SH-SY5Y细胞毒性损伤中的作用

目的研究胆碱能M受体(主要为M1受体)参与SH-SY5Y细胞周期依赖性蛋白激酶5(Cdk5)调控及其在敌敌畏(DDVP)诱导SH-SY5Y细胞毒性损伤中的作用。方法①应用免疫荧光结合共聚焦显微镜检测胆碱能M1受体及Cdk5在SH-SY5Y细胞中的分布和表达。②细胞对照组、氧化震颤素(Oxo-M)组(1×10^-4mol·L^-1作用48 h)、罗考唯亭(Rosc)组(培养结束前1 h加入Rosc 1×10^-4mol·L^-1)、Rosc+Oxo-M组(Rosc 1×10^-4mol·L^-1预处理1 h后,再加入Oxo-M 1×10^-4mol·L^-1作用至48 h),应用MTT比色法检测细胞存活率,Western印迹法检测Cdk5表达。③细胞对照组、DDVP组(1×10^-5mol·L^-1作用48 h)、Rosc组(培养结束前1 h加入1×10^-4mol·L<...

Acetyl cholinergic M receptor regulates cyclin dependent kinase 5 and plays pivotal role in DDVP induced cytotoxic injury in SH-SY5Y cells

OBJECTIVE To investigate the regulatory effect of muscarinic acetyl cholinergic M receptor(M1 subtype)on cyclin-dependent kinase 5(Cdk5)and the mechanism of neurotoxic injury induced by Cdk5-mediated dimethyl-dichlorovinyl-phosphate(DDVP)in SH-SY5Y cells.METHODS①The laser confocal was used to detect the expressions of M1 receptor and Cdk5 in SH-SY5Y cells.②The cell control,oxotremorine(Oxo-M)(Oxo-M 1×10-4 mol·L-1 was treated for 48 h),roscovitine(Rosc)(Rosc 1×10-4 mol·L-1 for 1 h),and Rosc+Oxo-M(Rosc 1×10-4 mol·L-1 pre-treated for 1 h followed by Oxo-M 1×10-4 mol·L-1 for 47 h)groups were detected by MTT assay to analyze cell viability while Western blotting used to evaluate the expression of Cdk5.③The cell control,DDVP(DDVP 1×10-5 mol·L-1 was treated for 48 h),Rosc(Rosc 1×10-4 mol·L-1 for 1 h),Rosc+DDVP(Rosc 1×10-4 mol·L-1 pre-treated for 1 h,followed by DDVP 1×10-5 mol·L-1 for 47 h)groups were detected by MTT assay to analyze cell viability,while Western blotting was used to evaluate the expression of Cdk5.AnnexinⅤ-FITC/PI staining and TUNEL staining were used to detect the rate of cell apoptosis.RESULTS①The results of laser confocal microscopy confirmed that M1 receptor and Cdk5 were expressed in SH-SY5Y cells.②When Oxo-M(1×10-4 mol·L-1)treated SH-SY5Y cells for 48 h,the cell survival rate was significantly reduced to(59.1±7.3)%,and the expression of Cdk5 was significantly increased(vs control,P<0.05).When Rosc 1×10-4 mol·L-1 treated SH-SY5Y cells for 1 h,the expression of Cdk5 was significantly increased(vs control,P<0.05).The cell viability of Rosc+Oxo-M significantly increased to(84.5±20.9)%,and the expression of Cdk5 was significantly decreased(vs Oxo-M,P<0.05).③When DDVP(1×10-5 mol·L-1)treated SH-SY5Y cells for 48 h,the cell survival rate was significantly reduced to(68.6±4.1)%,and the expression of Cdk5 was significantly increased(vs control,P<0.05).The cell viability of Rosc+DDVP significantly increased to(84.4±8.8)%,but the expression of Cdk5 was significantly decreased(vs DDVP,P<0.05).AnnexinⅤ-FITC/PI combined with TUNEL staining results showed that the apoptosis rate of DDVP increased to(64.1±8.4)%and TUNEL-positive cells were significantly increased(vs control,P<0.05),but the apoptosis rate of Rosc+DDVP was reduced to(34.4±9.5)%.Rosc significantly suppressed DDVP-induced TUNEL-positive cells of SH-SY5Y cells(vs DDVP,P<0.05).CONCLUSION The over-activation of cholinergic M receptor results in increasing Cdk5 activity as well as SH-SY5Y cytotoxicity.Furthermore,the enhancement of Cdk5 activity plays a critical role in DDVP induced injury of SH-SY5Y cells,and the inhibition of Cdk5 activity can significantly reduce the cytoxicity caused by DDVP and Oxo-M.